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Localization of the DAZ gene expression in seminiferous tubules of patients with spermatogenic disorders.  [cached]
Anna Szczerba,Anna Jankowska,Miros?aw Andrusiewicz,Jerzy B Warcho?
Folia Histochemica et Cytobiologica , 2006, DOI: 10.5603/4578
Abstract: The research on the expression and mutations of DAZ and its homologues in human and other mammals suggests that protein products of these genes can mainly affect development of germinal cells. The aim of the present study was to analyze the expression of the DAZ gene in seminiferous tubules of six men with spermatogenic disorders (hypospermatogenesis and spermatogenic arrest). The results based on the RT-PCR IS technique demonstrated that the DAZ product was present only in some seminiferous tubules and the fluorescence intensity was different within individual tubules. The most intense fluorescence characterised the spermatogonia, especially these organised in small groups inside separate tubules. In the patients with spermatogenic arrest at the spermatocyte stage, the DAZ gene transcripts were also found in primary spermatocytes. However, the fluorescence intensity of primary spermatocytes, except the fluorescence of the spermatocytes localised upon the lumen, was weaker than the fluorescence of spermatogonia. The results of our study showed that DAZ gene activity seems to correspond to the proliferative activity of stem cells of germinal epithelium.
Reconstruction of Mouse Testicular Cellular Microenvironments in Long-Term Seminiferous Tubule Culture  [PDF]
Juho-Antti M?kel?, Jorma Toppari, Adolfo Rivero-Müller, Sami Ventel?
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0090088
Abstract: Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilm's tumour antigen 1 (WT1) and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA) and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger), LIN28, Gpr125 (G protein-coupled receptor 125), CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs) that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH) which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2–4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to establish in vitro conditions that allow spontaneous reconstruction of testicular cellular microenvironments.
Localization of dynamin 2 in rat seminiferous tubules during the spermatogenic cycle.
Iguchi H,Watanabe M,Kamitani A,Nagai A
Acta Medica Okayama , 2002,
Abstract: Dynamin is a protein essential to endocytosis. Dynamin 2, a dynamin isoform, is expressed most intensely in testicular tissue; however, precise localization has never been studied. Therefore, we investigated the expression of dynamin 2 in rat testicular tissue using immunohistochemical methods, and discuss here the physiological function of this protein. Testicular tissues were obtained from Wistar rats at 10, 21 and 63 days of age. Immunohistochemistrical examination and Western blot analysis were conducted using dynamin 2 specific antibody. Western blot analysis showed that expression in 21- and 63-day-old rats was more intense than that in 10-day-old rats. Dynamin 2 expression was observed using immunohistochemical method in the seminiferous tubules of all rats. In the 63-day-old rats, the expression was intense, especially in spermatids in the earlier maturation stages and in spermatocytes, and was observed in Sertoli cells. However, in spermatids, the expression gradually declined as spermatids matured to spermatozoa. In the 21-day-old rats, the expression was evident in spermatocytes and Sertoli cells, but that in the 10-day-old rats was weak. Intense expression of dynamin 2 during spermatogenesis suggests that this protein plays an important role in this process.
The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse
Ke Zheng, Xin Wu, Klaus H Kaestner, Peijing Wang
BMC Developmental Biology , 2009, DOI: 10.1186/1471-213x-9-38
Abstract: In the present study, we show that LIN28, a pluripotency factor, is specifically expressed in undifferentiated spermatogonia (As, Apr, and Aal) in mouse. Ngn3 also specifically labels undifferentiated spermatogonia. We used Ngn3-GFP knockin mice, in which GFP expression is under the control of all Ngn3 transcription regulatory elements. Remarkably, Ngn3-GFP is only expressed in ~40% of LIN28-positive As (single) cells. The percentage of Ngn3-GFP-positive clusters increases dramatically with the chain length of interconnected spermatogonia.Our study demonstrates that LIN28 specifically marks undifferentiated spermatogonia in mice. These data, together with previous studies, suggest that the LIN28-expressing undifferentiated spermatogonia exist as two subpopulations: Ngn3-GFP-negative (high stem cell potential) and Ngn3-GFP-positive (high differentiation commitment). Furthermore, Ngn3-GFP-negative cells are found in chains of Ngn3-GFP-positive spermatogonia, suggesting that cells in the Aal spermatogonia could revert to a more primitive state.Spermatogenesis is a productive self-renewing system of adult stem cells that continuously generates spermatozoa through life. At the foundation of this system is the spermatogonial stem cells (SSCs) [1-4]. In mouse testis, isolated A (single) spermatogonia (As) are believed to be the most primitive cells and contain the stem cells. In normal situations, while half of As cells divide and give rise to Apr (paired) spermatogonia that are interconnected by cytoplasmic bridges due to incomplete cytokinesis, the remaining half of As cells undergo self-renewal divisions. The Apr spermatogonia further divide to become chains of 4, 8, 16, or 32 Aal (aligned) spermatogonia. The As, Apr, and Aal spermatogonia can only be identified by their topographical configurations on the basement membrane of the seminiferous tubules and are collectively referred to as "undifferentiated" spermatogonia, although this nomenclature causes confusion becaus
Cytokeratin 18 expression in immature Sertoli cells: co-localization with interstitial lymphocytic infiltrates.  [cached]
Roland Kruse,Sibylle Eigelshoven,Anna Kaiser,Thomas Ruzicka
Folia Histochemica et Cytobiologica , 2009, DOI: 10.5603/4371
Abstract: Although multiple interactions of seminiferous tubules and the interstitial testicular tissue are known, correlation of cytokeratin 18 expressing Sertoli cells with interstitial changes has still not yet been reported. Considering this fact, we focused our investigation on changes of the adjacent interstitial tissue. A total sample of 51 testicular biopsies (from infertile patients) showing mixed atrophy was examined immunohistochemically with antibodies against cytokeratin 18, vimentin, L26/CD20, CD4 and CD8. Twenty-one of the 51 cases showed single seminiferous tubules with Sertoli cells expressing cytokeratin 18. These 21 tubules consistently exhibit either spermatogenic arrest at the level of spermatogonia or only immature Sertoli cells. In the adjacent interstitial tissue of 8 of the 21 cytokeratin 18 positive tubules (39%) striking inflammatory infiltrates--predominantly expressing L26/CD20 typical for B lymphocytes and CD8 typical for T suppressor lymphocytes--were detected. These findings underline that tubules with cytokeratin 18 expressing Sertoli cells exhibit early spermatogenic arrest or only few remaining Sertoli cells. Additionally, we observed a remarkable co-localization of these tubules with lymphocytic infiltrates of the adjacent interstitial tissue.
The Ubiquitin C-Terminal Hydrolase L-1 (PGP9.5) as a Suitable Marker to Trace and Isolate Spermatogonia Cells from Ovine Testis
Uyunbilig Borjigin,Sachula Wu,Zhiying He,Xin Wang,Muren Herrid,Bou Shorgan
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2012.4364.4368
Abstract: The Ubiquitin C-terminal Hydrolase L-1 (UCH-L1) is usually named as PGP9.5. Earlier study indicated that PGP9.5 has been used to identify type A spermatogonia in some species such as mouse and pig. In the present study, the localization of PGP9.5 was studied on paraffin sections of the ram testis samples in order to understand the correlation between gonocyte migration and spermatogonia location in the seminiferous tubules of ovine testis development. The results also indicated that testes from neonatal ovine had only one type of undifferentiated germ cell gonocyte and PGP9.5 as a spermatogonia cell marker was suitable for isolation and enrichment of ovine Spermatogonial Stem Cells (SSCs). Thus, the finding will be helpful for the future study on ovine SSCs in long-term culture and transplantation.
Changes in the Seminiferous Epithelium of the Testes during Postnatal Development in Assam Goat  [PDF]
Kamal Sarma,J. Devi
Anatomy Research International , 2012, DOI: 10.1155/2012/620924
Abstract: The present work is conducted to elucidate the postnatal development of the seminiferous epithelium of the testes of the Assam goats from 0 day to 10 months of age. A total of eighteen Assam goats divided into six age groups, namely, group-I (0-day), group-II (2 months), group-III (4 months), group-IV (6 months), group-V (8 months), and group-VI (10 months), consisting of 3 animals in each group were used in this study. The seminiferous tubules did not have lumina up to the age of 2 months, hence called the sex cords, and these contained centrally placed gonocytes and peripherally located sustentacular cells. Initiation of spermatogenesis started in 4-month old kids. Luminization process was completed by 6 months of age with all the seminiferous tubuyes having well-developed lumina at this age. These seminiferous tubules contained all the spermatogenic cells of the adult testis. Onset of puberty was observed to be established at 6 months of age in the Assam goats as evidenced by presence of spermatozoa adhering to the adluminal border of the Sertoli cells as well as in the tubular lumen. The histomorphology of various cells of the seminiferous epithelium has been described. 1. Introduction India possesses 122.92 millions of goats of which 29.06 lacs are found in Assam [1]. Goat rearing has tremendous potential in the northeastern states particularly among the small and marginal farmers and landless laborers because of very low initial investment and adequate financial returns. More than 85 percent of the population in this region is nonvegetarian, and chevon is preferred by all as it has no religious taboo. Postnatal anatomical studies on the male genital system at various ages, particularly the testis and its tubular system, are important to know the anatomical growth and development. A few anatomical studies on testes are reported in goats, namely, biometry of the testes in the Sirohi goats [2], testicular measurements in the Assam local X Beetal goats [3], and testicular growth in the British Saanen, Alpine, and Toggenburg breeds of bucks [4]. Some works were also conducted in other ruminants elucidating morphology and biometry of the testes such as buffalo [5] and rams [6]. This study is the first report for the postnatal development of the seminiferous epithelium in the Assam goat. 2. Materials and Methods A total of 18 male Assam goats varying in age from 0-day to 10 months were used in the present study. The animals were divided into six age groups, namely group-I (0-day), group-II (2 months), group-III (4 months), group-IV (6 months), group-V (8
Morphological study of the testes of the dove Columba livia (Gmelin) (Columbidae, Columbiformes)
Maruch, Sandra Maria das Gra?as;Teles, Maria Eloiza de Oliveira;Ribeiro, Maria das Gra?as;
Revista Brasileira de Zoologia , 1995, DOI: 10.1590/S0101-81751995000100015
Abstract: known as "domestic dove", the columba livia (gmelin, 1789) is a columbidae species widely distributed in brazil, whose reproductive biology has been studied by many researchers. the testes of 12 columba livia males were collected and prepared for histologic examination under an optical microscope, the results of which were analysed and photographed. the tunica albuginea that covers the testes consists of a thick, not very cellular layer of dense connective tissue. groups of interstitial cells with typical morphological appearance and surrounded by loose, well vascularized connective tissue are observable within the organ, between the seminiferous tubules. the seminiferous tubules are thick, intensely wound and, when seen in cross section, show sertoli cells and spermatogenic lineage cells in different stages of development. these include spermatogonia (type a, clear; type a, dark; and type b), spermatocytes i and ii, spermatids, and a large number of spermatozoons. similarities are found between the histological findings described and those reported for the testes of columbina talpacoti (temminck, 1811).
Histomorphological Alterations in the Prostate Gland and Epithelium of Seminiferous Tubule of Sprague-Dawley Rats Treated with Methanolic Extract of Momordica charantia Seeds
Yama Oshiozokhai Eboetse,Duru Francis Ikechukwu,Okanlawon Abayomi Olugbenga,Oremosu Ademola Ayodele
Iranian Journal of Medical Sciences , 2011,
Abstract: Background: There is yet a dearth of literature on the antifertility effect of Momordica charantia on the male reproductive system. The aim of this study was to determine the effect of graded oral doses of methanolic seed extract of Momordica charantia on the histology of prostate gland and seminiferous tubules of rats. Methods: Forty male Sprague-Dawley rats, weighing 176±7 g were assigned randomly into four main groups A to D of 10 rats per group. Groups A to C received daily oral doses of15, 25 or 50 mg/100 g body weight of the seed extract for 56 days. Group D (control) received physiological saline. In each group, five rats were sacrificed on day 57, the remaining half on day 113 (56 days after withdrawal of the extract). The testes and prostate were processed for histological examination. Results: There was a dose-related alteration in the cytoarchitecture of seminiferous tubules with marked reduction in spermatogenic series. The prostate gland showed dilatation as well as increased intraluminal secretions with increasing dose. Moreover, there was a significant recovery of prostate tissue as the sections were similar to their control counterpart. Conclusion: the findings of the present study indicate that methanolic extract of Momordica charantia seeds caused reversible histological alterations in the prostate and testes of Sprague-Dawley rats.
Structural alterations in the seminiferous tubules of rats treated with immunosuppressor tacrolimus
Breno H Caneguim, Paulo S Cerri, Luís C Spolidório, Sandra M Miraglia, Estela Sasso-Cerri
Reproductive Biology and Endocrinology , 2009, DOI: 10.1186/1477-7827-7-19
Abstract: Rats aging 42-day-old received daily subcutaneous injections of 1 mg/kg/day of tacrolimus during 30 (T-30) and 60 (T-60) days; the rats from control groups (C-30 and C-60) received saline solution. The left testes were fixed in 4% formaldehyde and embedded in glycol methacrylate for morphological and morphometric analyses while right testes were fixed in Bouin's liquid and embedded in paraffin for detection of cell death by the TUNEL method. The epithelial and total tubular areas as well as the stages of the seminiferous epithelium and the number of spermatocytes, spermatids and Sertoli cells (SC) per tubule were obtained.In the treated groups, seminiferous tubules irregularly outlined showed disarranged cellular layers and loss of germ cells probably due to cell death, which was revealed by TUNEL method. In addition to germ cells, structural alterations in the SC and folding of the peritubular tissue were usually observed. The morphometric results revealed significant decrease in the number of SC, spermatocytes, spermatids and significant reduction in the epithelial and total tubular areas.Tacrolimus induces significant histopathological disorders in the seminiferous tubules, resulting in spermatogenic damage and reduction in the number of Sertoli cells. A careful evaluation of the peritubular components will be necessary to clarify if these alterations are related to the effect of FK-506 on the peritubular tissue.The immunosuppressive therapy in organ transplant recipients has included the use of calcineurin inhibitors such as tacrolimus (FK-506) and cyclosporine [1]. Although the mechanism of action of tacrolimus is similar to that of cyclosporine, tacrolimus is 10 to 100 times more potent and has been referred as useful therapeutic option for the transplant recipients. Tacrolimus is a macrolide immunosuppressant isolated from fermentation broth of Streptomyces tsukubaensis [2]. This drug binds to a specific immunophilin, called FKBP12, forming a complex in the l
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